Cystic fibrosis (CF) is an autosomal recessive disorder characterized by abnormalities in water and electrolyte transport that lead to pancreatic and pulmonary insufficiency. Taussig, L. M., An overview. In Cystic Fibrosis. L. M. Taussig, ed. (New York: Thieme-Stralton), 1-9 (1984). It is one of the most common severe autosomal recessive disorders, having a 5% carrier frequency and affecting about 1 in 2500 live births in North America.
Functional expression of the CF defect reduces the chloride ion permeability of epithelial tissues. Quinton, P. M., Faseb J. 4, 2709-2717 (1990). The ability of epithelial cells in the airways, sweat glands, pancreas and other tissues to secrete CI in response to cAMP-mediated agonists is most or severely reduced. Activation of apical membrane Cl channels by cAMP-dependent protein dinase (PKA) is impaired, but channels with normal conductance properties can be activated by other means, including agonists whose effects are mediated by increased cell Ca. Frizzell, R. A. et al., Trends Neurosci, 10, 190-194 (1987); Welsh, M. J., FASEB J. 4, 2718-2725 (1990). These finds suggest that the Cl channel per se is not defective in CF, but that the defect might lie in a regulatory protein that transduces the effects of protein kinase activation. The presence of abnormalities in epithelial sodium transport in CF cells further supports the concept of a regulatory defect that can affect other cellular functions. Boucher, R. C. et al., J. Clin. Invest. 78, 1245-1252 (1986).
Isolation of the gene for CF, as described in detail in the aforementioned related applications has provided further insight into the molecular basis of the disease. See also Rommens, J. M. et al., Science 245, 1059-1065 (1989); Riordan, J. R. et al., Science 245, 1066-1073 (2989); Derem, B. S. et al., Science 245, 1073-1080 (1989). The gene responsible for CF has been localized to 250,000 bp of genomic DNA based on its location within the genome. This gene encodes a protein of 1480 amino acids called the cystic fibrosis transmembrane conductance regulator (CFTR). Riordan et al., supra.
The most compelling evidence thus far to support the role of CFTR in the etiology of CF has been provided by genetic analyses. Kerem et al., supra, (1989). Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of mutations, including nonsense and frameshift mutations. Cutting, G. R. et al., Nature 346, 366-369 (1990); White, M. B. et al., Nature 344, 655-667 (1990); Dean, M. et al., Cell 16, 863-870 (1990); Kerem, B. S. et al., identification of mutations in regions corresponding to the 2 putative nucleotide (ATP) binding folds of the cystic fibrosis gene, PNAS (USA) (1990) (in press). However, extensive population studies have indicated that the most common CF mutation is a deletion of the three nucleotides that encode phenylalanine 508 (.DELTA.F.sub.508). This deletion is present on 70% of all CF chromosomes, but not on normal chromosomes. Kerem et al., supra (1989); The Cystic Fibrosis Genetic Analysis Consortium (1990).
Results from both physiological and molecular cloning studies have raised the possibility that CFTR is a Cl channel. The defect in Cl channel activation by cAMP-dependent protein kinase (PKA) is present at the single-channel level in cell-free membrane patches and the protein structure predicted from CF gene closing suggests that CFTR is an integral membrane protein with twelve membrane-spanning domains. Schoumacher, R. A. et al., Nature 330, 152-754 (1987); Li, M. et al., Nature 331, 358-360 (1988); Riordan et al., supra. The identification of CF-associated alterations in other cellular processes such as amiloride-sensitive Na transport and mucin sulfation also supports the view that CFTR may regulate several cellular processes. Boucher et al., supra; Boat, T. F. et al; Arch. Biochem. Biophys. 17, 95-104 (1976).
Although the specific role that CFTR plays in Cl transport remains to be determined, the CFTR protein contains several interesting functional domains including two nucleotide binding folds, a regulatory region that has many possible sites for phosphorylation, and two hydrophobic regions that probably interacted with cell membranes. CFTR shows structural similarity with several members of the "ATP binding cassette" (ABC) superfamily of proteins, including the periplasmic binding proteins of prokaryotes, and the P-glycoprotein associated with multidrug resistance in higher eudaryotes. Riordan et al., supra; Hyde, S. C. et al., Nature 346, 312-365 (1990).
Recent progress in our understanding of the genetic and functional basis of CF has provided a foundation for better defining its molecular pathology as well as developing novel therapies based on somatic gene transfer.